The Bacillus subtilis conjugative transposon ICEBs1 mobilizes plasmids lacking dedicated mobilization functions

Abstract

Integrative and conjugative elements (ICEs, also known as conjugative transposons) are mobile elements that are found integrated in a host genome and can excise and transfer to recipient cells via conjugation. ICEs and conjugative plasmids are found in many bacteria and are important agents of horizontal gene transfer and microbial evolution. Conjugative elements are capable of self-transfer and also capable of mobilizing other DNA elements that are not able to self-transfer. Plasmids that can be mobilized by conjugative elements are generally thought to contain an origin of transfer (oriT), from which mobilization initiates, and to encode a mobilization protein (Mob, a relaxase) that nicks a site in oriT and covalently attaches to the DNA to be transferred. Plasmids that do not have both an oriT and a cognate mob are thought to be nonmobilizable. We found that Bacillus subtilis carrying the integrative and conjugative element ICEBs1 can transfer three different plasmids to recipient bacteria at high frequencies. Strikingly, these plasmids do not have dedicated mobilization-oriT functions. Plasmid mobilization required conjugation proteins of ICEBs1, including the putative coupling protein. In contrast, plasmid mobilization did not require the ICEBs1 conjugative relaxase or cotransfer of ICEBs1, indicating that the putative coupling protein likely interacts with the plasmid replicative relaxase and directly targets the plasmid DNA to the ICEBs1 conjugation apparatus. These results blur the current categorization of mobilizable and nonmobilizable plasmids and indicate that conjugative elements play a role in horizontal gene transfer even more significant than previously recognized.

Fig 1

Map of ICEBs1 and various mutants. (A) Genetic map of ICEBs1. ICEBs1 is shown in its linear integrated form. Open arrows indicate open reading frames and the direction of transcription. Gene names are indicated above the arrows. The origin of transfer (oriT) is indicated by a thick black line overlapping the 3′ end of conQ and the 5′ end of nicK. oriT is needed for ICEBs1 transfer (34) and replication (33). The small rectangles at the ends of ICEBs1 represent the 60-bp direct repeats that contain the site-specific recombination sites in the left and right attachment sites, attL and attR, that are required for excision of the element from the chromosome. (B to E) Schematic diagram of deletion mutations in ICEBs1 in donor strains. Thin horizontal lines represent the regions of ICEBs1 that are present in the donor strains. Gaps in the line represent the regions of ICEBs1 that are deleted. Antibiotic resistance markers that are inserted in some of the alleles are not shown. Δ(rapI-phrI) (B, D, and E) (5) and Δ(conG-yddM) (C) (34) are deletion-insertion mutations with a kanamycin resistance gene inserted (not shown). ΔnicK (D) and ΔconQ (E) are unmarked in-frame deletion mutations that do not disrupt the expression of downstream genes and do not disrupt oriT. (F to I) Schematic diagram of truncated ICEBs1 derivatives integrated at thrC. These constructs cannot excise due to deletion of attR and were used to provide ICEBs1 genes in trans for complementation tests of the ICEBs1 ΔconQ mutant. Thin horizontal lines represent the regions of ICEBs1 that are present at thrC. (B to I) The abilities or inabilities of plasmids to be mobilized or of ICEBs1 to be transferred from various donor strains are summarized as + or −, respectively. nd, not determined.

Fig 2

Plasmid maps. Schematic diagrams of plasmids pC194 (A), pBS42 (B), and pHP13 (C), all mobilized by ICEBs1. The approximate size of each plasmid is indicated under the plasmid name. Circles represent each plasmid and are not shown to scale. Thin black arrows and gene names within each circle represent functional genes or, in the case of ′mobU (B), a nonfunctional 3′ fragment of mobU (missing the 5′ end). Antibiotic resistance genes include cat (chloramphenicol resistance) and mls (macrolide, lincosamide, and streptogramin resistance). Small open arrows on each circle represent the origin of replication, which contains the site for nicking by the cognate replicative relaxase (encoded by rep, repU, and rep60). The sequences flanking the nic sites in each plasmid ori are highly conserved. The orientation of the open arrows indicates the direction of leading strand synthesis for rolling circle replication. pBS42 (B) and pHP13 (C) are composites of segments from several plasmids (6, 23). The junctions and sources of the different segments are indicated by the intersecting lines and plasmid names on the perimeter of each circle. For pBS42 (B), the locations of two NsiI restriction sites are indicated with a line and an “N.” The three plasmids have a 0.98-kb sequence, containing cat, in common. Other DNA sequences common to all three plasmids are ≤12 bp.